Polyketides are a large family of structurally diverse natural products with a broad range of biological activities, including antibiotic and pharmacological properties. They are synthesized by multifunctional polyketide synthases (PKSs), which catalyze the successive decarboxylative condensation of acetate, propionate or butyrate coenzyme- A thioesters in a process analogous to fatty-acid biosynthesis. Although their structures and mechanism of action are poorly understood, PKSs show great promise (or use in the metabolic engineering of organisms to produce novel, biologically-active products. Heterologous expression and genetic engineering of PKSs have resulted in the engineered biosynthesis of more than twenty novel polyketides in the Khosla Lab; however, the current understanding of basic aspects of PKS biochemistry limits the extent to which rational engineering can be performed. The goal of the research described in this proposal is to broaden the understanding of PKSs so that they may be used more effectively in the production of novel polyketides. Efforts will be directed toward the purification and in vitro reconstitution of components of a type II polyketide synthase. Although many mechanistic aspects of PKSs remain to be explored, the major efforts of this work will be directed toward understanding aspects of PKS enzymology that are unique to PKSs, such as its oligomerization state and the mechanism of polyketide chain elongation.